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Processing apoferritin with the Appion pipeline.

Scott M Stagg1, Joshua H Mendez2

  • 1Institute of Molecular Biophysics, 91 Chieftain Way, Florida State University, Tallahassee, FL 32306, United States; Department of Chemistry and Biochemistry, 95 Chieftain Way, Florida State University, Tallahassee, FL 32306, United States.

Journal of Structural Biology
|July 4, 2018
PubMed
Summary
This summary is machine-generated.

This study tested cryo-electron microscopy (cryo-EM) workflows for 3D reconstruction. Maximizing particle numbers, after removing poor-quality data, improved apoferritin map resolution, demonstrating an effective strategy for high-quality cryo-EM analysis.

Keywords:
Best practicesCryo-EMMap challengeSingle particle reconstruction

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Area of Science:

  • Structural Biology
  • Biophysics
  • Computational Biology

Background:

  • Single particle cryo-electron microscopy (cryo-EM) is a powerful technique for determining high-resolution structures of biological macromolecules.
  • The 3DEM map challenge aimed to evaluate different algorithms and workflows for cryo-EM data processing.
  • Apoferritin, a protein complex, presents unique challenges in single particle reconstruction.

Purpose of the Study:

  • To assess the efficacy of a standard Appion workflow with minimal manual intervention for cryo-EM data processing.
  • To investigate the impact of particle sorting and elimination on the resolution and quality of 3D reconstructions.
  • To optimize the cryo-EM reconstruction strategy for the apoferritin dataset.

Main Methods:

  • Utilized the standard Appion workflow for processing single particle cryo-EM data.
  • Focused on the apoferritin dataset, known for reconstruction challenges.
  • Compared reconstructions generated using different numbers of particles (25,844 vs. 53,334) and evaluated the impact of particle selection and mask creation parameters.

Main Results:

  • Achieved high-resolution 3D maps (3.7 Å and 3.6 Å) for apoferritin, ranking among the best submitted maps.
  • Demonstrated that including a larger number of particles, after rigorous junk elimination, led to improved resolution.
  • Showcased that subtle changes in custom mask creation parameters significantly influenced the final 3D reconstruction resolution.

Conclusions:

  • For conformationally homogeneous particles like apoferritin, maximizing particle usage post-cleaning is crucial for achieving highest resolution.
  • The choice of parameters for mask creation in cryo-EM data processing can subtly yet significantly impact resolution.
  • The standard Appion workflow, with careful parameter selection, can yield competitive results in cryo-EM map challenges.